Resting-state fMRI (R-fMRI) is a major method for studying human brain networks. It relies on correlations in spontaneous fluctuations of BOLD signal. However BOLD signal fluctuations are poorly understood, leading to ambiguity in interpretation of R-fMRI derived networks. For task fMRI (T-fMRI) we, and others, have shown a direct relationship between BOLD signal changes of glutamatergic neuronal signaling and oxidative demand. The evoked changes in oxidative demand (CMRO2), measured by calibrated T-fMRI combines measurements of BOLD signal, blood flow (CBF), blood volume (CBV), and subject-specific biophysical constants. However a similar level of metabolic understanding for R-fMRI has been elusive. In the past cycle, using calibrated T-fMRI with 1H[13C] MRS and extracellular recordings, we developed and validated transfer functions of BOLD, CBF, CBV, and CMRO2 signals that related them directly to extracellular recordings of multi-unit activity (MUA) and local field potentials (LFP). Inverse of the transfer functions allowed calibrated T-fMRI maps to be converted into neuronal activity maps. However this approach has limitations when applied to calibrated R-fMRI, in particular the small spatial scale of MUA/LFP recordings is not suitable for correlating with large regions involved in correlated network fluctuations and lack of a stimulus trigger in resting-state to allow recordings in and out of scanner to be time synchronized. We will overcome these limitations by simultaneous calibrated R-fMRI and calcium (Ca2+) imaging in Snap25-GCaMP6 mice, which contain genetically encoded fluorescent Ca2+ reporters. Since Ca2+ imaging directly measures neuronal activity in these transgenic mice, we will improve the biomarker potential of R-fMRI derived functional connectivity density (FCD) differences in health and disease. Preliminary data show that the spatiotemporal structure of R-fMRI and resting Ca2+ (R-Ca2+) networks quite similarly, and long-term mitochondrial health (e.g., aging) can also perturb R-fMRI network patterns. Since we, and others, have shown that the resting-state fluctuations depend on the total activity, an independent and absolute measure of total activity (by high-resolution 1H[13C] spectroscopic imaging) is critical, so that we can bridge the gap between BOLD signal and underlying activities of neuronal populations (by electrophysiology). We build on these preliminary results and propose: In Aim 1 we will develop technologies for simultaneous calibrated R-fMRI and R-Ca2+ imaging. In Aim 2 we will measure and validate state-dependent neurovascular and neurometabolic transfer functions. In Aim 3 we will apply calibrated R-fMRI and R-Ca2+ imaging in aging, with and without calorie restriction,to validate that the methods can accurately track how neuronal resting-state fluctuations are altered and sensitivity to an intervention. Given that network changes as revealed by fMRI- derived FCD is a feature of brain disorders (e.g., autism, schizophrenia, healthy aging) calibrating R- fMRI to R-Ca2+ could help interpret the altered network dynamics that underlie disease.